Figure 5. IFC analysis of erythrocyte invasion by AMA1^KO merozoites.

(a) WT GFP⁺ merozoites isolated from blood cultures were incubated with PKH26-stained erythrocytes in normal conditions (top panels) or with 1 μM cytochalasin D (bottom panels). Left panels: plots of GFP intensity in the erythrocyte population. Cells associated with a merozoite (GFP⁺) are gated as Ery^A (green gate), and invaded cells are gated as Ery^I using an algorithm recognizing merozoites associated with a tight-fitting PV (blue gate). (b) PKH26-stained erythrocytes non-invaded (top) or invaded by a merozoite surrounded by the duplication of PKH26 upon PV formation (bottom). A plot of the mean grey value of PKH26 (red) and GFP (green) signals per pixel is shown on the right of each image. The white arrows show the path of measured pixels. Scale bars, 2 μm. (c) Quantification of Ery^A (green) and Ery^I (blue) populations in the assay described in a represented as percentages of parasites from initial input. Results are mean±s.d. from three independent experiments. (d) Ery^A (green) and Ery^I (blue) scored for WT or AMA1^KO merozoites isolated from blood cultures and incubated with erythrocytes for 10 min. Data show mean±s.d. of triplicates, representative of two independent experiments. (e) Ery^A (green) and Ery^I (blue) scored for WT or AMA1^KO merozoites isolated from blood cultures and incubated with erythrocytes for 3 min. Data show the mean of two independent experiments. KO, knockout.